Methods and compositions for promoting survival and proliferation of endothelial cells and stimulating angiogensis

ABSTRACT

The present invention relates to adenovirus E4ORF1 gene and to endothelial cells engineered to express the E4ORF1 gene. The present invention also relates to uses of the E4ORF1 gene, and cells expressing the E4ORF1 gene, and to compositions comprising the E4ORF1 gene, or comprising cells expressing the E4ORF1 gene.

This application is a continuation of U.S. application Ser. No.13/897,829, filed on May 20, 2013, which is a continuation of U.S.application Ser. No. 12/523,372, filed on Dec. 22, 2009, now U.S. Pat.No. 8,465,732, which was a national stage application under 35 U.S.C. §371 of International Application Serial No. PCT/US2008/51499, filed onJan. 18, 2008, which claims the benefit of priority to U.S. ApplicationSer. No. 60/881,667, filed on Jan. 22, 2007, and U.S. Application Ser.No. 60/881,225, filed on Jan. 19, 2007. Each of the foregoingapplications is incorporated by reference into the present application.

This invention was supported, in part, by NIH grant R01-HL075234.Therefore, the U.S. government has certain rights in this invention. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

A portion of the disclosure of this patent document contains materialthat is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure, as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in an ASCII text file, named as 30171ZY_SEQ.txt of2 KB, created on Feb. 26, 2018, and submitted to the United StatesPatent and Trademark Office via EFS-Web, is incorporated herein byreference.

BACKGROUND OF THE INVENTION

The adenoviral early 4 (E4) region contains at least 6 open readingframes (E4ORFs). The entire E4 region has been shown to regulateangiogenesis and promote survival, but not proliferation, of endothelialcells (see, for example, Zhang et al. (2004), J. Biol. Chem.279(12):11760-66). Prior to the present invention, it was not knownwhether all of the ORFs within the E4 region, a subset of the E4ORFs, ora single specific E4ORF, might be responsible for this activity. Use ofthe entire E4 region, either clinically or experimentally, to induceangiogenesis or to promote survival or proliferation of endothelialcells, may not be desirable because some of the E4ORFs can havedeleterious effects. For example, the E4ORF6 gene is known to induceapoptosis (Yamano et al. (1999) J. Virol. 73:10095-103). Also, theE4ORFs are immunogenic and therefore administration of all of the E4ORFsto subjects may not be desirable. Accordingly, there was a need in theart to identify the sequences within the E4ORF region responsible forits pro-angiogenic and pro-endothelial cell survival effects. Thepresent invention solves this problem in the art by identifyingsequences within the E4ORF region that are useful for inducingangiogenesis and for promoting survival and proliferation of endothelialcells.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a method for expandingendothelial cells (ECs) in culture, comprising obtaining a sample ofprimary endothelial cells, introducing into the primary endothelialcells a nucleic acid molecule comprising the adenovirus E4ORF1 geneunder the control of a suitable promoter to produce E4ORF1-expressingendothelial cells, and culturing the E4ORF1-expressing endothelialcells. The step of “introducing” can be performed by any suitablemethod, such as by transfection or by viral-mediated transduction. In anembodiment, lentivirus-mediated transduction is used. In certainembodiments, the cells can be cultured in the absence of serum or growthfactors.

In another embodiment, the present invention provides a method forinducing angiogenesis in a subject by administering endothelial cellsexpressing the E4ORF1 gene. For example, the present invention providesa method for inducing angiogenesis comprising obtaining a sample ofprimary endothelial cells, introducing into the primary endothelialcells a nucleic acid molecule comprising the adenovirus E4ORF1 geneunder the control of a suitable promoter to produce E4ORF1-expressingendothelial cells, and administering the E4ORF1-expressing endothelialcells to the subject. In certain embodiments, the promoter can be aninducible promoter. The subject can be any subject in which it isdesired to induce angiogenesis. In an embodiment, the subject is amammal. In another embodiment, the subject is a human. The subject canbe suffering from, or at risk of developing, an ischemic condition, suchas for example, myocardial ischemia. The subject can also have a wound,such that the subject would benefit from angiogenesis during woundhealing. In some embodiments, the present invention provides autologoustransplantation methods whereby endothelial cells are obtained from thesubject, are engineered to express the E4ORF1 gene, and are thenre-administered to that same subject in order to induce angiogenesis.

In certain embodiments, the present invention also provides “genetherapy” methods. For example, the present invention provides a methodfor inducing angiogenesis in a subject by administering a compositioncomprising a nucleic acid encoding the adenovirus E4ORF1 gene under thecontrol of a suitable promoter. Such methods can be useful for, interalia, the treatment of ischemic conditions and for wound healing.

The present invention also provides populations and cultures ofendothelial cells expressing the adenovirus E4ORF1 gene, andcompositions containing such cells, such as therapeutic compositions.Endothelial cells are present in all tissues and any source of cells canbe used in accordance with the present invention. Examples ofendothelial cells that can be used in the present invention, include,but are not limited to, endothelial cells derived from the testis, lung,lymphatic tissue, cord blood, umbilical vein, heart, skin, liver, brain,bone, and pancreas.

In another embodiment, the present invention provides methods fortargeting therapeutic agents to tumors by administeringE4ORF1-expressing endothelial cells conjugated to a therapeutic agent toa subject having a tumor. Such cells can incorporate into the newlyforming tumor vasculature and therefore provide a means of selectivelytargeting therapeutic agents to the site of the tumor. Examples oftherapeutic agents that can be used include, but are not limited to,chemotherapeutic agents, toxins, and radionuclides. In certainembodiments, the therapeutic agent is conjugated to theE4ORF1-expressing endothelial cells using an antibody. The presentinvention also provides methods for targeting therapeutic proteins orpeptides to a tumor by administering E4ORF1-expressing endothelial cellsthat also express a heterologous nucleotide sequence that encodes aprotein or peptide that has anti-tumor activity.

In another embodiment, the present invention provides methods forproducing cultures of endothelial cells suitable for use as feeder cellsto support the growth of stem or progenitor cells. In one embodimentsuch a method comprises obtaining a sample of primary endothelial cells,introducing into the primary endothelial cells a nucleic acid moleculecomprising the adenovirus E4ORF1 gene under the control of a suitablepromoter, and culturing the E4ORF1-expressing endothelial cells. Thepresent invention also provides feeder cell cultures comprisingE4ORF1-expressing endothelial cells. Such cultures are particularlyuseful for supporting the growth of stem or progenitor cells in culture.Stem and progenitor cells that can be supported by E4ORF1-expressingendothelial feeder cells can be derived from any source from where stemand progenitor cells can be harvested.

In yet another embodiment, the present invention provides methods forproducing cultures of ECs suitable for use as feeder cells to supportthe growth of primary cancer cells. In vitro studies on primary cancercells have been difficult to conduct due to the inherent difficulty ofprimary cancer cells to grow in culture and due to their unknown growthfactor requirements (Drexler et al. (2003) Leukemia 17:416-26).Accordingly, in one embodiment, E4ORF1-expressing endothelial cells areused as feeder cells to support the growth of primary cancer cells inculture in the absence of serum or growth factors. Examples of primarycancer cells that can be supported by the E4ORF1-expressing endothelialcells of the present invention include, but are not limited to, breast,colon, prostate, liver, lung, bone, epithelial, glial, neuronal, kidney,testis, ovarian, and pancreatic cells.

In another embodiment, the present invention provides methods forstudying organ-specific endothelial cells and tumor-derived endothelialcells. In particular, the present invention provides a model forinvestigating angiogenesis and neovascularization events related toorganogenesis, tumorigenesis, and metastasis in serum-free,cytokine-free conditions in vitro. In one embodiment, such a methodprovides obtaining a sample of organ-specific endothelial cells,introducing into the organ-specific endothelial cells a nucleic acidmolecule comprising the adenovirus E4ORF1 gene under the control of asuitable promoter, and culturing the E4ORF1-expressing organ-specificendothelial cells. In another embodiment, the method comprisesintroducing a nucleic acid molecule comprising the adenovirus E4ORF1gene under the control of a suitable promoter into tumor-derivedendothelial cells and culturing said tumor-derived endothelial cells.The present invention also provides for an in vivo method ofinvestigating angiogenesis and neovascularization events related toorganogenesis and tumorigenesis. Specifically, the method comprisesinfecting endothelial cells or tumor-derived endothelial cells withlentivirus packaged to express E4ORF1, expanding the E4ORF1-expressingendothelial or tumor-derived endothelial cells in serum-free,cytokine-free culturing media, mixing the E4ORF1-expressing endothelialor tumor-derived endothelial cells with Matrigel™, and injecting theMatrigel™-endothelial cell mixture into the flanks of mice.

The present invention further provides populations and cultures ofendothelial progenitor cells (EPCs) expressing the adenovirus E4ORF1gene and compositions containing such cells, such as therapeuticcompositions. Due to their diverse differentiation potential, EPCs canbe quite useful in the field of vascular regenerative therapy in anumber of different tissues. Expression of E4ORF1 in EPCs can thus avoidthe need to isolate and culture endothelial cells from specific tissuesources. The ability to isolate and culture EPCs is known. See, forexample, Ingram et al. (2005) Blood 106:1525-31 or Ingram et al. (2004)Blood 104:2752-60.

The present invention further teaches that E4ORF1 sequences from orderived from other adenovirus types or strains can also be used topromote the survival and proliferation of ECs according to the methodsand compositions disclosed herein. Because all E4ORF1 proteins possesscellular growth-transforming ability when expressed at a certain level(Tauber and Dobner (2001) Oncogene 20:7847-54; Weiss et al. (1997) JVirol 71:1857-70), their ability to promote the survival andproliferation of ECs and EPCs should be conserved as well. Accordingly,in one embodiment, ECs or EPCs expressing the E4ORF1 of human adenovirus5 can be used as disclosed herein. In another embodiment, the E4ORF1 ofhuman adenovirus 9 can be used. The methods and compositions describedcan be modified to use the E4ORF1 sequence from any adenovirus type orstrain.

These and other embodiments of the invention are described further inthe accompanying written description, examples, drawings, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-F provide data showing that AdE4ORF1 prolongs survival ofendothelial cells (ECs). In FIG. 1A, human umbilical vein endothelialcells (HUVEC) monolayers were cultured in serum-free, growth factor-freemedium and treated with AdE4ORF1→7 (which carries the full complement ofE4 complex genes), ADE4ORF1 (which expresses only E4ORF1),AdE4ORF3,4,6,6/7 (which lacks expression of E4ORF1), AdE4ORF4 (whichexpresses only E4ORF4) and AdE4ORF6 (which expresses only E4ORF6). Theviability of ECs was quantified 3 days after infection. Data arepresented as means±S.E. from three independent experiments. *, differsfrom control, p<0.01. Phase-contrast microscopy depicting cellmorphology (FIG. 1B). HUVEC monolayers were infected with either PBScontrol, Lenti-E4ORF1, or Lenti-GFP for 3 days in serum-free, growthfactor-free medium (FIG. 1C). FIG. 1D is a depiction of the number ofHUVECs after infection with Lenti-E4ORF1 or Lenti-GFP for the indicatedtime. Lymphatic endothelial cells were infected with either PBS control,Lenti-E4ORF1, or Lenti-GFP for 6 days in serum-free, growth factor-freemedium (FIG. 1E). In FIG. 1F, enzymatically digested human testiculartissue and human cord blood mononuclear cells were infected withLenti-E4ORF1 for 1 day in endothelial growth medium and subsequentlyexpanded for several passages.

FIGS. 2A-E demonstrates that E4ORF1 activates pAkt in endothelial cells(ECs). In FIG. 2A, HUVEC monolayers were infected with AdE4ORF3,4,6,6/7at 20 MOI, AdE4ORF1 at 20 MOI, and AdE4 vectors at 100 MOI inserum-free, growth factor-free medium for 48 hours. The cell lysateswere then analyzed by immunoblot using polyclonal anti-phospho-Ser473Akt antibody (pAkt) and total Akt antibody (Akt). In FIG. 2B, ECs weretransfected with 20 nM E4ORF1 siRNA, E4ORF6 siRNA or the controlfluorescence siRNA for 24 hours, then ECs were exposed to AdE4 vectors(100 m.o.i) in serum-free, growth factor-free medium for 2 days.Expression of pAkt or Akt was determined by Western blot usingphospho-specific anti-phospho-Akt antibody (pAkt) or anti-total Aktantibody (Akt). ECs were infected with Lenti-E4ORF1 or Lenti-GFP for 3days; expression of pAkt or Akt was determined by Western blot (FIG.2C). In FIG. 2D, ECs were infected with Lenti-E4ORF1 or Lenti-GFP ascontrol. After 3 days, the Lenti-E4ORF1-transfected cells were incubatedin the presence or absence of rapamycin (50 ng/ml) with or withoutLY-294002 (LY) (10 μg/ml) for 3 days in serum-free, growth factor-freemedium. A viable cell count was taken using the trypan blue exclusionassay. Data are presented as means±S.E. from three independentexperiments. Lenti-E4ORF1 infected-ECs were treated with medium alone ormedium supplemented with 10 μM PD98059 (PD), or SB203580 (SB) for 2days. The viability of ECs in serum-free, growth factor-free medium wasquantified (FIG. 2E).

FIGS. 3A-F provide data that demonstrates that AdE4ORF1-mediatedactivation of the FGF-2/FGF-receptor pathway promotes the survival andproliferation of endothelial cells (ECs). In FIG. 3A, HUVEC monolayerswere infected with AdE4ORF1, AdE4ORF3,4,6,6/7 at 20 MOI, or AdE4ORF1→7,AdE4^(Null) at 100 MOI in serum-free, growth factor-free medium for 48h. The cell lysates were then analyzed by immunoblot using polyclonalanti-FGF2 antibody and anti-β-Actin antibody. FIG. 3B shows ECs infectedwith Lenti-E4ORF1 for 24 hours, then transfected with siRNA againstFGF-2 for 48 hours. The cell lysates were then analyzed by immunoblotusing polyclonal anti-FGF2 antibody and anti-β-Actin antibody. In FIG.3C, ECs were infected with Lenti-E4ORF1 for 24 hours, then transfectedwith siRNA against FGF-2 for 48 hours. After three days in culture inserum-free, growth factor-free medium, the number of proliferating ECswas determined by trypan blue exclusion. FIG. 3D is a phase contrastmicroscopy picture of AdE4ORF1-infected ECs treated with control siRNAor FGF-2 siRNA. In FIG. 3E, Lenti-E4ORF1 infected-ECs were treated with10 μM SU5402, a FGFR inhibitor or 10 μM SU5416, a VEGFR2 inhibitor for48 hours in serum-free, growth factor-free medium or serum-free, FGF-2supplemented medium. After 2 days in culture, the number of viable cellswas counted. The time course of proliferation of naïve (control) andAdE4ORF1-infected ECs was assessed in serum-free, growth factor-freeconditions, as well as serum-free medium supplemented with FGF-2 (5ng/ml) and/or VEGF-A (10 ng/ml). The number of viable cells was countedevery two to four days and plotted (FIG. 3F).

FIGS. 4A-H show in vivo survival and angiogenesis of ECs in NOD/SCIDmice. Mice received subcutaneous inoculations of Matrigel™ containingeither human EC alone or AdE4ORF1-expressing ECs. In the absence ofE4ORF1, human ECs survived poorly after 5 days, forming barelydetectable masses (FIGS. 4A and 4C), whereas ECs expressing E4ORF1formed obvious hemorrhagic lesions, both at the gross level (FIG. 4B) orin histologic sections stained with hematoxylin and eosin (FIG. 4D)Immunohistochemistry staining for human endothelium (anti-humanVE-cadherin) revealed strong staining in areas containing human ECsexpressing E4ORF1 (FIG. 4E), whereas mouse endothelium (labeled withMECA-32) was detectable only adjacent to the EC masses (FIG. 4F, arrowsindicate mouse vessels). In FIG. 4G, abundant FGF-2 expression waspresent in E4ORF1-expressing human EC. In FIG. 4H, presence ofanti-phospho-Akt staining in the nuclei of E4ORF1-expressing ECs wasconsistent with the in vitro results.

FIGS. 5A-I show that E4ORF1-expressing endothelial cells (ECs) arecapable of tubulogenesis, sprouting and supporting leukemic cell growth.Mice received subcutaneous inoculation of 10×10⁶ GFP-expressing ECs(control) (FIGS. 5A and 5C) or ECs expressing both GFP and E4ORF1 (FIGS.5B, 5D and 5E). The mice were administered intravenously humanspecific-UEA-1 lectin just prior to sacrifice 14 days after subcutaneousinoculation of the ECs. Cryosections were stained to detect UEA-1 as ameasure of vessels that were functional at the time of sacrifice. FIGS.5A and 5B depict single confocal slices through areas containingGFP-positive vessel-like structures. FIGS. 5C, 5D and 5E depict fullthickness projections through areas containing functional donor-derivedhuman vessels. In FIGS. 5F, 5G, and 5H, HL60 tumor cells wereco-inoculated with E4ORF1-expressing ECs subcutaneously and grown 21days prior to harvest. Occasional vessel-like structures with a lumenwere labeled by anti-human VE-cadherin (FIG. 5F) within the HL60 tumorsthat had been co-inoculated with E4ORF1-expressing ECs, although thevast majority of vessels were MECA-32 positive and of host origin (FIG.5G). FIG. 5H shows little co-localization of mouse and human markers inchloromas but confirmed the specificity of the antibodies via confocalmicroscopy. In FIG. 5I, HL60 cells were cultured in serum-free, growthfactor-free medium in the presence or absence of E4ORF1-expressingendothelial cells and counted.

FIG. 6 provides a graph showing that a feeder cell layer ofE4ORF1-transduced endothelial cells induced expansion of CD34+hematopoetic progenitor cells grown on this feeder cell over a five weekperiod even in the absence of serum. The number of cells is indicated onX axis. The various cytokine treatments are indicated on the Y axis.“x-vivo” represents the basal medium, “FGF2” refers to mediumsupplemented with basic fibroblast growth factor, “5% FBS” refers tomedium supplemented with 5% fetal bovine serum, and “GM” refers tomedium supplemented with granulocyte monocyte colony stimulating factor.

FIG. 7 sets forth the E4ORF1 nucleotide sequence of human adenovirus 5,which was used in Example 1 (SEQ ID NO:1).

FIGS. 8A-D show that testicular cells expressing E4ORF1characteristically expressed endothelial cell markers. Testicular biopsytissue was enzymatically dissociated, transduced at passage zero withE4ORF1, and serially passaged and expanded using standard EC growthmedium. Homogenous cells with endothelial morphology were stained withvon Willebrand factor (FIG. 8A), VE-cadherin (FIG. 8B), CD34 (FIG. 8C),and CD31 (FIG. 8D) along with prodium iodide.

FIGS. 9A-C show that E4ORF1 stimulates migration and tube formation ofECs. FIG. 9A demonstrates the ability of AdE4ORF1 to induce migration ofendothelial cells in a wound healing assay. HUVECs were infected withAdE4ORF1 (20 MOI) for 24 hours, then the EC monolayers were wounded witha pipette chip and incubated in the serum-free, growth factor-freemedium. After wounding of uninfected ECs, ECs were treated with orwithout FGF-2 (10 ng/ml) in serum-free, growth factor-free medium, asparallel control. ECs were photographed at 6 hours, 24 hours or 48 hoursafter wound healing. Each experiment was performed in triplicate and atleast 3 times. FIG. 9B shows that AdE4ORF1-expressing cells support tubeformation. ECs were infected with AdE4ORF3,4,6,6/7, AdE4ORF1, or PBS(control) and plated on Matrigel™-coated culture plates for 8 hours andanalyzed for typical neo-angiogenic tube formation by phase-contrastmicroscopy. In FIG. 9C, ECs were infected with Lenti-GFP or Lenti-E4ORF1in serum-free, growth factor-free medium for two days and observed underphase-contrast microscopy for their ability to form neo-angiogenic tubes8 hours after plating on Matrigel™.

FIG. 10 identifies the common vascular EC markers expressed by bothE4ORF1-expressing primary endothelial cells (PECs) and naïve PECs.

DETAILED DESCRIPTION

The present invention relates to the adenovirus E4ORF1 gene and toendothelial cells engineered to express the E4ORF1 gene. The presentinvention also relates to uses of the E4ORF1 gene, and uses of cellsexpressing the E4ORF1 gene, and also relates to compositions and cellscomprising the E4ORF1 gene. The summary of the invention, the examples,and the claims describe some of the embodiments of the invention.Further description of certain aspects of the invention is providedbelow.

E4ORF1

The present invention is based, in part, upon the discovery that theE4ORF1 gene within the larger E4 region (which also encodes multipleother ORFs) has certain biological effects on endothelial cells, such aspromoting survival and inducing proliferation of endothelial cells andalso stimulating angiogenesis. In certain embodiments, the E4ORF1 geneused is the whole adenovirus E4ORF1 gene, or a variant, mutant orfragment thereof that has the functional properties described herein.The sequence of the human adenovirus type 5 E4 region (containing ORF1)is available on Genbank (see for example accession number D12587). Inone embodiment of the invention, the E4ORF1 sequence used is thatprovided in FIG. 7 (SEQ ID NO: 1) or a sequence with greater than 85%sequence identity to SEQ ID NO:1. In another embodiment, the variant ormutant of the E4ORF1 sequence is a sequence with about an 85% identityto SEQ ID NO:1, or about an 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to SEQ ID NO:1 that retains theability to induce angiogenesis or promote the survival and proliferationof ECs. In another embodiment, a fragment of the E4ORF1 is a sequencewhich varies in length by ±30 nucleotides relative to SEQ ID NO:1, orabout ±28 nucleotides, ±26 nucleotides, ±24 nucleotides, ±22nucleotides, ±20 nucleotides, ±18 nucleotides, ±16 nucleotides, ±14nucleotides, ±12 nucleotides, ±10 nucleotides, ±9 nucleotides, ±8nucleotides, ±7 nucleotides, ±6 nucleotides, ±5 nucleotides, ±4nucleotides, ±3 nucleotides, ±2 nucleotides, or ±1 nucleotides relativeto SEQ ID NO:1, all of which retain the properties described herein,including, but not limited to, the ability to induce angiogenesis andthe ability to promote survival and proliferation of endothelial cells.

Alternatively, the E4ORF1 sequence used can be, or can be derived from,other adenoviruses types or strains. Examples of other adenoviral E4ORF1sequences include, but are not limited to, human adenovirus 9 (GenbankAccession No. CAI05991), human adenovirus 7 (Genbank Accession No.AAR89977), human adenovirus 46 (Genbank Accession No. AAX70946), humanadenovirus 52 (Genbank Accession No. ABK35065), human adenovirus 34(Genbank Accession No. AAW33508), human adenovirus 14 (Genbank AccessionNo. AAW33146), human adenovirus 50 (Genbank Accession No. AAW33554),human adenovirus 2 (Genbank Accession No. AP_000196), human adenovirus12 (Genbank Accession No. AP_000141), human adenovirus 35 (GenbankAccession No. AP_000607), human adenovirus 7 (Genbank Accession No.AP_000570), human adenovirus 1 (Genbank Accession No. AP_000533), humanadenovirus 11 (Genbank Accession No. AP_000474), and human adenovirus 3(Genbank Accession No. ABB17792).

In some embodiments the E4ORF1 gene can be used in conjunction with oneor more other genes or gene fragments from the E4 region, such as theE4ORF2, E4ORF3, E4ORF4, E4ORF5 and/or E4ORF6 genes, or variants, mutantsor fragments thereof. For example, the E4ORF1 region can be used inconjunction with one or more other genes or gene fragments from the E4region for the production of E4ORF1-expressing endothelial feeder cells.However, in other embodiments, the E4ORF1 gene is not in the context ofthe entire E4 region, or not in the context of the E4ORF2, E4ORF3,E4ORF4, E4ORF5 and/or E4ORF6 regions. For example, although the E4ORF1gene can be used in a construct (such as a viral vector) that containsother genes or other coding regions (such as marker genes, antibioticresistance genes, and the like), in certain embodiments, the E4ORF1 geneis not used in a construct that contains the entire E4 region or thatcontains other ORFs from the E4 region, such as E4ORF2, E4ORF3, E4ORF4,E4ORF5 and/or E4ORF6.

The E4ORF1 gene can be used in constructs that contain various othergenes or coding regions, depending on the desired use. For example, theE4ORF1 gene can be used in conjunction with antibiotic resistance genes,reporter genes or expression tags (such as, for example, GFP), or anyother genes that might be desirable to express in endothelial cells. TheE4ORF1 gene can also be expressed as part of a fusion protein. TheE4ORF1 gene can also be used in conjunction with any desired gene,coding region, or indeed non-coding regions, that may be present in theexpression construct or viral vector used, or any other desired gene,coding region, or non-coding regions that is desired.

The E4ORF1 gene can be under the control of a promoter to allow forexpression. Any promoter able to drive expression of the E4ORF1 gene inthe desired cell type can be used. Examples of suitable promotersinclude, but are not limited to, the CMV, SV40, RSV, HIV-Ltr, and MMLpromoters. The promoter can also be a promoter from the adenovirusgenome, or a variant thereof. For example, the promoter can be thepromoter used to drive expression of E4ORF1 in an adenovirus.

In some embodiments, the E4ORF1 gene can be placed under the control ofan inducible promoter, so that expression of the E4ORF1 gene can beturned on or off as desired. Any suitable inducible expression systemcan be used, such as for example, a tetracycline inducible expressionsystem or a hormone inducible expression system. This can be useful forin vivo applications. For example, the E4ORF1 gene can be expressedwhile it is needed to promote angiogenesis and then switched off whenthere has been sufficient angiogenesis for the desired outcome, forexample when there has been sufficient angiogenesis to treat an ischemiccondition or heal a wound. The ability to turn off expression of theE4ORF1 gene would be useful for ensuring that there is not an excessiveamount of angiogenesis or an excessive amount of endothelial cellproliferation in vivo.

Any suitable means of transfecting or transducing endothelial cells withthe E4ORF1 gene can be used. For example, the E4ORF1 gene can betransfected into cells using standard methods known in the art,including, but not limited to, liposome-mediated transfection,polybrene-mediated transfection, DEAE dextran-mediated transfection,electroporation, calcium phosphate precipitation, microinjection, ormicro-particle bombardment. Similarly, the E4ORF1 gene can be deliveredto endothelial cells using a viral delivery system such as lentivirus,adenovirus, retrovirus, adeno-associated virus or herpesvirus deliverysystem. In an embodiment, the E4ORF1 gene is delivered to endothelialcells using a lentiviral gene delivery system.

The present invention also provides vectors, including expressionvectors and viral vectors, which contain the E4ORF1 sequence, preferablyunaccompanied by other adenovirus E4ORFs such as E4ORFs 2-6. In otherembodiments, the present invention provides a lentivirus vectorcomprising the E4ORF1 sequence, preferably unaccompanied by otheradenovirus E4ORFs such as E4ORFs 2-6. In another embodiment, the E4ORF1sequence used in the vector is SEQ ID NO:1, or any variant, mutant orfragment thereof that retains the properties described herein,including, but not limited to, the ability to induce angiogenesis andthe ability to promote survival and proliferation of endothelial cells.Such vectors can be useful for, inter alia, transfecting or transducingendothelial cells in vitro or in vivo.

Subjects

As used herein, the term “subject” is used to refer to any animal orhuman. In one embodiment, the subject is a mammal selected from thegroup consisting of primates (such as humans and monkeys), rodents,(such as mice, rats and rabbits), ovine species (such as sheep andgoats), bovine species (such as cows), porcine species, equine species,feline species and canine species. Another embodiment of the inventionis where the subject is a human. In certain embodiments the subject issuffering from, or at risk of developing an ischemic condition, such asmyocardial ischemia, or some other condition that would benefit from orbe alleviated by increased angiogenesis, for example wound healing.

Methods of Culturing and Preserving E4ORF1-Expressing Endothelial Cells

The present invention provides methods for culturing E4ORF1-expressingendothelial cells. Once endothelial cells have been transfected ortransformed with the E4ORF1 gene, the cells have increased proliferativecapacity, increased survival capacity and reduced senescence. Thus, theE4ORF1-expressing cells are suitable for long-term culture andexpansion. The cell cultures can also be cryopreserved. One of skill inthe art can readily culture and cryopreserve the E4ORF1-expressingendothelial cells of the invention using methods known to those skilledin the art, such as the methods described in Culture of Animal Cells: AManual of Basic Technique, 4th Edition (2000) by R. Ian Freshney(“Freshney”), the contents of which are hereby incorporated byreference, and also using the methods described in the Examples.

Feeder Cells Expressing E4ORF1

Stem and progenitor cells are notoriously difficult to maintain and growin culture. Many such cells must be cultured with a “feeder cell” layeror grown in medium conditioned by such feeder cells. However, finding asuitable source of feeder cells that will support stem cell growth, thatare free from contamination, and which can be propagated underconditions that are also suitable for propagation of stem cells, isdifficult. For example, in many situations primary cultures of cells,such as primary cultures of endothelial cells are used as feeder cells.However, feeder layers of primary cells, such as endothelial cells,often require the presence of serum or growth factors for maintenance.This can be problematic, as many stem cells cannot tolerate the presenceof serum or certain growth factors because, for example, stem cells willdifferentiate in the presence of such factors. Also, many primary celltypes used as feeders are not proliferative, or have low proliferativecapacity, meaning that new primary cultures need to be generatedfrequently. This is time consuming and also leads to problems ofinconsistency between batches of feeder cells. It would be desirable tohave a source of feeder cells that support stem and progenitor cellgrowth and that are proliferative, have minimal requirements in terms ofserum and growth factors, and which can be used reliably as a consistentand reproducible source of feeder cells.

E4ORF1-expressing endothelial cells of this invention are particularlyuseful as feeder cells for use in methods for culturing stem cells. Dataprovided in Example 2 illustrates the suitability of these cells asfeeders to support the growth of stem cells, in particular hematopoieticstem cells and progenitor cells. The E4ORF1-expressing endothelial cellsof the invention can also be useful as feeder cells for other types ofstem or progenitor cells, including, but not limited to, embryonic stemcells and stem cells derived from any fetal or adult tissues such ashematopoietic stem cells, neural stem cells, skin stem cells,spermatogonial stem cells, gut stem cells, cancer stem cells and thelike.

The E4ORF1-expressing cells of the invention can be plated in a layer onthe surface of a culture vessel and stem cells or progenitor cells, orcell populations believed to contain stem or progenitor cells can beplated on top of the E4ORF1-expressing “feeder layer.” TheE4ORF1-expressing feeder cells can also be used as a source ofconditioned medium for the culture of stem or progenitor cells, or canbe used in conjunction with other feeder cell/stem cell culturetechniques known and used in the art. The invention further provides fora composition which comprises conditioned media obtained from culturingthe E4ORF1-expressing cells of the present invention.

For similar reasons as stem and progenitor cells, primary cancer cellsare also notoriously difficult to grow and maintain in culture withoutimmortalization. Accordingly, it would be desirable to have a source offeeder cells that support primary cancer cell growth, have minimalrequirements in terms of serum and growth factors, and which can be usedreliably as a consistent and reproducible source of feeder cells. TheE4ORF1-expressing endothelial cells of the present invention can also beused as feeder cells for use in methods for culturing primary cancercells. The E4ORF1-expressing cells of the invention can be plated in alayer on the surface of a culture vessel and primary cancer cells can beplated on top of the E4ORF1-expressing “feeder layer.” TheE4ORF1-expressing feeder cells can also be used as a source ofconditioned medium for the culture of primary cancer cells, or can beused in conjunction with other feeder cell/cancer cell culturetechniques known and used in the art. The invention further provides fora composition which comprises conditioned media obtained from culturingthe E4ORF1-expressing cells of the present invention.

Therapeutic Compositions Comprising E4ORF1-Expressing Endothelial Cells

Several embodiments of the invention involve therapeutic compositionscomprising E4ORF1-expressing endothelial cells. These compositionscomprise a preparation of E4ORF1-expressing endothelial cells, asdescribed above, and a carrier solution suitable for administration toliving subjects, such as humans. In one embodiment, the carrier solutionis a physiological saline solution. Other therapeutically acceptableagents can be included if desired. One of ordinary skill in the art canreadily select suitable agents to be included in the therapeuticcompositions depending on the desired outcome.

Methods of Treatment Using E4ORF1-Expressing Endothelial Cells

The present invention also provides various methods for inducingangiogenesis in subjects, and methods for treating conditions such asischemic conditions, by administering the E4ORF1-expressing endothelialcells of the invention to the subject. One of ordinary skill in the artcan readily perform such treatment methods by preparing a therapeuticcomposition containing E4ORF1-expressing endothelial cells, andadministering the therapeutic composition to a suitable subject.

The cells can be administered to subjects using any suitable means knownin the art. For example, the cells can be administered by injection orinfusion into the blood stream at a location peripheral to the sitewhere the cells are needed, or by injection or infusion into the bloodstream in the vicinity of the region where the cells are needed, or bydirect infusion or injection into tissue, either at the site where thecells are needed, or in the vicinity of the site where the cells areneeded, or at a peripheral location. In the case of treatment ofmyocardial ischemia, the cells are administered directly to, or in thevicinity of, the heart. In the case of treatment of wounds, the cellsare administered directly into, or in the vicinity of, the site of thewound, for example the skin in the case of a skin wound. The cells canbe administered in a single dose or in multiple doses. The skilledartisan will be able to select a suitable method of administrationaccording to the desired use.

The present invention teaches that primary or tumor-derived endothelialcells expressing the adenoviral E4ORF1 gene are angiogenic even in theabsence of serum or growth factors. In addition, the present inventionalso teaches that expression of E4ORF1 induces FGF-2 expression.Accordingly, the cells of the present invention can be used to produceFGF-2 where FGF-2 is needed, without introducing the E4ORF1-expressingendothelial cells which can undergo angiogenesis where angiogenesis isnot wanted. Thus, another embodiment entails administeringE4ORF1-expressing endothelial cells in an environment (such as within amatrix, a device, a sheath or artificial layer, or within a barrier)wherein the E4ORF1-expressing endothelial cells are immobilized andcannot migrate, while allowing FGF-2 to escape and act upon cellsneeding FGF-2. In another embodiment of the invention, various growth orinhibitory factors secreted by primary endothelial cells, tumor-derivedendothelial cells or endothelial progenitor cells can be used tolocalize delivery of these growth or inhibitory factors. Examples ofgrowth or inhibitory factors secreted by these cells include, but arenot limited to, FGFs, PDGF, insulin, erythropoietin, VEGF, TGF-β, G-CSF,GM-CSF, NGF, EGF, or LIF. For example, one of ordinary skill in the artcan envision microencapsulating E4ORF1-expressing endothelial cells toachieve this purpose. Alternatively, a dialysis bag can be used. In anycase, one of ordinary skill in the art can readily identify and employmethods well-known in the art that allow for the growth factors to besecreted while retaining the cells.

Methods of Drug Targeting

In certain embodiments, the present invention provides a method oftargeting certain agents to tumors in subjects by administering to thesubject E4ORF1-expressing endothelial cells that have been engineeredfor delivery of such agents. Because tumors frequently stimulate thein-growth of new blood vessels into the tumor (stimulate tumorangiogenesis), E4ORF1-expressing endothelial cells delivered to asubject can contribute to the new tumor vasculature. Thus, the cells canbe used to deliver agents directly to a tumor site. Examples of agentsthat can be targeted to tumors using E4ORF1-expressing endothelial cellsinclude, but are not limited to, cytotoxic drugs, other toxins,prodrugs, radionuclides, and gene expression products. For example,E4ORF1-expressing endothelial cells can be engineered such that theyalso express a protein having anti-tumor activity, or such that theysecrete, release, or are coated with a toxic agent such as achemotherapeutic agent or radionuclide. For example, radionuclide drugsor chemotherapeutic drugs can be conjugated to an antibody that binds tothe surface of the E4ORF1-expressing endothelial cells and thereby usedto deliver the radionuclides or chemotherapeutic drugs to a tumor.Tumors can also be targeted using E4ORF1-expressing endothelial cellsengineered to secrete a prodrug-activating enzyme. After administrationof the non-toxic prodrug, the E4ORF1-expressing ECs that also expressingthe prodrug enzyme can convert the prodrug into its toxic form in thesecells.

These and other embodiments of the invention are further described inthe following non-limiting examples.

EXAMPLES Example 1 Effects of Adenovirus E4ORF1 Gene on EndothelialCells and on Angiogenesis

The adenoviral early 4 region (E4) contains at least 6 open readingframes (E4ORFs) and has been shown to promote survival of endothelialcells (ECs) and regulate angiogenesis. This invention provides thatadenoviral vectors expressing E4ORF1 (AdE4ORF1), but not vectors with anE4ORF1 deletion (e.g., AdE4ORF1^(null), AdE4ORF4 or AdE4ORF6), promotedEC survival in the absence of serum and pro-angiogenic growth factors.E4ORF1 expression using a lentiviral vector has a similar effect. Theknockdown of E4ORF1 by siRNA targeted against E4ORF1 mRNA suppressedsurvival of ECs infected with either AdE4ORF1 or vectors expressing thefull complement of adenoviral E4 (AdE4ORF 1→7). This invention providesthat AdE4ORF1 and Lenti-E4ORF1 induced Akt activation via Ser473phosphorylation and increased FGF-2 synthesis in ECs. Knocking downFGF-2 expression by siRNA abrogated the E4ORF1 EC survival effect.

Furthermore, the migration and capillary-tube formation wassignificantly enhanced in AdE4ORF1-infected ECs. Subcutaneousadministration of Lenti-E4ORF1-infected ECs in mice inducedneovascularization. These results indicate that the adenoviral E4ORF1gene product exerts a pro-angiogenic effect by promoting survival andmigratory potential of endothelial cells, and can explain the vasculartoxicity observed during gene therapy clinical trials.

The gene products encoded by the early region 4 of adenovirus vectors(AdE4) not only code for key proteins essential for virus replicationbut also appear to be involved in the regulation of transcription,post-translational modifications, cell cycle, apoptosis, DNA repair andcell signaling (Tauber and Dobner (2001) Oncogene 20(54):7847-54;Leppard (1997) J Gen Virol 78 (Pt 9):2131-8). AdE4 promotes angiogenesisby modulating the migration, apoptosis and inflammatory potential ofendothelial cells (Ramalingam et al. (1999) Blood 93(9):2936-44; Rafiiet al. (2001) Circ Res 88(9):903-10; Zhang et al. (2004) J Biol Chem279(12):11760-66; Zhang et al. (2005) Circ Res 96(9):950-7). AdE4promotes EC survival via regulation of several key intracellularsignaling molecules, by increasing Src kinase and phosphatidylinositol3-kinase (PI3K) phosphorylation and the Bcl2/Bax ratio, as well asreducing caspase-3 activity (Ramalingam et al. (1999) Blood93(9):2936-44; Zhang et al. (2004) J Biol Chem 279(12):11760-66). AdE4also regulates the gene expression of connexins 40 and 43 in ECs andmouse heart tissue (Zhang et al. (2005) Circ Res 96(9):950-7). However,the identity of genes transcribed by AdE4 that regulates angiogenesis isnot known.

AdE4 mRNA contains at least six open reading frames (E4ORFs), suggestingthat E4 encodes at least six gene products (Leppard (1997) J Gen Virol78 (Pt 9):2131-8); Bridge and Ketner (1989) J Virol 63(2):631-8). Amongthe known E4ORF genes, E4ORF1 has been shown to activate severalsignaling pathways that support cell survival. For example, E4ORF1 hasbeen shown to activate PI3K through a novel PDZ protein-dependentmechanism of action (Frese et al. (2003) Oncogene 22(5):710-21). Inaddition, E4ORF1 expression mimics growth factor signaling via mTOR byactivating PI3K (O'Shea et al. (2005) EMBO J 24(6):1211-21). Therefore,E4ORF1 expressed in a vector can support angiogenesis.

The present invention provides that infection by adenoviral vectorsexpressing E4ORF1 alone (AdE4ORF1) is sufficient to mimic the effects ofvectors expressing the full complement of adenoviral E4 (AdE4ORF1→7) onECs, prolonging cell survival in serum-free and growth factor-freeculture conditions. Specifically, AdE4ORF1 promotes angiogenesis viaactivation of PI3K-Akt signaling and induces fibroblast growth factor-2(FGF-2) production, thereby enhancing the survival and migratorycapacity of the ECs. The invention provides that expression of theAdE4ORF1 gene product is essential for the AdE4-mediated pro-survivaland pro-angiogenic effects on ECs.

Cell Culture.

Human umbilical vein endothelial cells (HUVECs) were isolated andcultured in EC medium (M199 medium containing 10% (v/v) fetal bovineserum, 20 μg/ml EC growth factor, 20 units/ml heparin, 100 μg/mlpenicillin and 100 μg/ml streptomycin) in a humidified incubator at 37°C. with air/5% CO₂ (Zhang et al. (2004) J Biol Chem 279(12):11760-66).HUVEC monolayers between passages 2 to 4 were used in these studies.Cell viability was assayed by the trypan blue exclusion method,indicating that fewer than 5% of the cells took up the dye both beforeand after the infection of adenoviral vectors.

Construction of Adenoviral Vectors.

The adenoviral vectors used included: AdE4ORF1→7, derived fromadenovirus type 5, which expresses E4ORFs from E4ORF1 to E4ORF6/7 buthas deletions of the E3 and E1 gene complexes with no transgene in theexpression cassette (Hersh et al. (1995) Gene Ther 2(2):124-31);AdE4ORF6 (expresses only E4ORF6 from the E4 promoter—all other E4ORFswere deleted); AdE4ORF1, which carries only E4ORF1 and has deletions ofE4ORFs 2, 3, 4, 5, 6/7; AdE4ORF3,4,6,6/7, which carries E4ORFs 3, 4, 6and 6/7, and has deletions of E4ORF1 and E4ORF2; AdE4ORF4 which carriesonly E4ORF4 and deletions of all other E4ORFs, and AdE4^(null) whichlacks the expression of all E4ORFs, as previously described (Querido etal. (1997) J Virol 71(5):3788-3798; Bridge and Ketner (1990) Virology174(2):345-353). The AdE4ORF4, AdE4ORF3,4,6,6/7, and AdE4ORF1 virusvectors were propagated on W162, a Vero-derived, E4-complementing cellline. AdE4ORF1→7 and AdE4ORF6 were amplified in 293 cells and purifiedby cesium chloride centrifugation and dialysis as described in Crystalet al. (1994) Nat Genet 8(1):42-51. All adenoviral vectors had aparticle/pfu ratio of approximately 100.

LentiE4ORF1—Generation of Lentivirus and Infection.

E4ORF1 gene was cloned into the Lentivirus vector. Lentiviruses weregenerated by co-transfecting 15 μg of lentiviral vector, 3 μg ofpENV/VSV-G, 5 μg of pRRE and 2.5 μg of pRSV-REV (see Dull et al. (1998)J Virol 72:8462-71) in 293T cells (passage 8-10, subconfluent) bycalcium precipitation method. Medium was changed 24 hours aftertransfection, and supernatants were collected 40 hours and 64 hoursafter transfection. Supernatants were immediately sterile-filtered usingsurfactant-free cellulose acetate membranes, aliquoted and stored at−80° C.

siRNA Preparation and Transfection.

siRNA for adenovirus E4ORF1 (target sequence,5′-GAAUCAACCUGAUGUGUUU-dTdT-3′) (SEQ ID NO:2) and E4ORF6 (targetsequence, 5′-GCCAAACGCUCAUCAGUGAUA-dTdT-3′) (SEQ ID NO:3), and FGF-2(target sequence, 5′-ACCCUCACAUCAAGCUACAACUUCA-dTdT-3′) (SEQ ID NO:4)were designed and synthesized by Invitrogen (Stealth™ RNAi). dTdT 3′overhangs were not part of the siRNA sequence, but rather were presentto facilitate RNA interference using short interfering RNA sequences.Twenty nM of siRNA preparation were transfected individually into ECsusing Lipofectamine™ 2000 following Invitrogen's protocols andexperiments were conducted 24 hours after transfection. The controlsiRNA (scrambled sequence), and siRNA against GFP, as another control,were used as the same concentrations. The transfection efficiency ofeach duplex siRNA (˜80%) was confirmed by using Block-iT™ FluorescentOligo (Invitrogen).

Western Blot Analysis.

Cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.1%sodium dodecyl sulfate, and 2 μg/ml aprotinin). Insoluble debris waspelleted, and the protein concentration of the supernatant wasdetermined with a DC protein assay kit (Bio-Rad). Fifty micrograms ofeach protein sample were separated on 10% SDS-PAGE gels. The proteinsamples were then transferred to nitrocellulose membrane. Proteinexpression was confirmed by immunoblotting with the followingantibodies: Ser473-phospho-Akt, total Akt, FGF-2, β-actin. Afterincubation with the appropriate primary and horseradishperoxidase-conjugated secondary antibodies, the membranes were developedwith enhanced chemiluminescence reagent (Amersham PharmaciaBiotechnology).

Wound Healing Assay.

HUVECs were maintained in subconfluence in EC medium, and then infectedwith AdE4ORF1, AdE4ORF3,4,6,6/7, or PBS. Twenty-four hours afterinfection, confluent HUVEC monolayers were wounded with a pipette chipand incubated in the serum-free, growth factor-free medium (X-vivomedium) for experiments. After 24 and 48 hours, the cells that hadmigrated across the edge of the wound were observed under microscopicvision. Each experiment was performed in triplicate and at least 3times.

Tube Formation Assay. The formation of vascular-like structures by HUVECon Matrigel™ (Becton Dickinson) was semi-quantified by phase contrastmicroscopy. Twenty-four well culture plates were coated with Matrigel™according to the manufacturer's instructions. HUVECs were infected withor without adenovirus, then seeded on coated plates at 5×10⁴ cell/wellin serum-free medium (SFM) and incubated at 37° C. for 8 hours.Uninfected HUVECs were seeded on coated plate in SFM containing VEGF-A(10 ng/ml) and FGF-2 (10 ng/ml) as a positive control.

In Vivo Matrigel™ Plug Assay.

To determine whether human E4ORF1-expressing endothelial cells canparticipate in neo-angiogenesis in vivo, these cells were inoculatedwith Matrigel™ plugs and/or HL60 leukemic cells. To assess angiogenesisin vivo, ECs were infected with Lenti-E4ORF1 and cultured for 7-14 days.Uninfected ECs were used as controls. Subsequently, ECs were detachedusing collagenase. 4×10⁶ ECs per mouse were mixed with Matrigel™ (BDBiosciences, San Jose, Calif.). The Matrigel™-EC mixtures (400 μl) wereimplanted into the flanks of two groups of mice. The mice weresacrificed after 5 to 10 days for analysis, at which time the Matrigel™plugs were photographed, removed and snap-frozen immediately in OCT(Tissue-Tek).

For functional analysis of the angiogenic potential of E4ORF1-expressingECs in vivo, ECs were co-infected with Lenti-E4ORF1 and Lenti-GFP in 35mm dishes and expanded. ECs infected with Lenti-GFP and passaged inparallel served as controls. Subsequently, ECs were detached usingcollagenase and 10×10⁶ ECs were mixed with 200 μl Matrigel™. TheMatrigel™-EC mixtures were subcutaneously injected into the flanks ofNOD-SCID mice (200 μl/flank). After two weeks, the mice wereanesthetized and inoculated intravenously with 100 μg ofbiotinylated-Ulex europaeus agglutinin-1 (UEA-1) (Sigma-Alderich, St.Louis, Mo.) to detect the incorporation of human ECs into vesselscontinuous with the mouse circulation just prior to sacrifice. After 30minutes, the mice were further anesthetized and transcardially perfusedwith 15 ml of 4% paraformaldehyde/PBS after initial flushing with 20 mlPBS. The Matrigel™ plugs were dissected out, post-fixed for 2-3 hours in4% paraformaldehyde/PBS, washed extensively, equilibrated for 48 hoursin 30% sucrose, and snap-frozen in OCT. To assess for functionalGFP-positive vessels, 30 micron cryosections were cut, blocked in 10%donkey serum for 30 minutes, and incubated with streptavidin-Alexa647conjugate (Invitrogen) for 30 minutes to detect biotinylated-UEA-1 boundto functional human vasculature. Slides were washed and mounted inglycerol containing 1 μg/ml propidium iodide (Sigma Alderich) as anuclear counterstain and examined by confocal microscopy using a Zeiss510 Meta confocal microscope for the presence of vessels co-stained forGFP and UEA-1.

In separate groups of mice, 5×10⁶ ECs were combined with HL60 leukemiacells. The Matrigel™-EC or Leukemia-EC mixtures were then injectedsubcutaneously into the flanks of the separate groups of NOD-SCID mice(400 μl/mouse). After 21 days, the mice were sacrificed and analyzed asdescribed above.

Immunostaining.

The presence of E4ORF1-expressing ECs was assessed using mouse and humanantibodies. Rabbit polyclonal anti-human FGF-2 antibody (Santa CruzBiotechnology, Santa Cruz, Calif.), rabbit polyclonal anti-phospho-Akt(Santa Cruz Biotechnology, Santa Cruz, Calif.), goat polyclonalanti-human VE-cadherin 1 antibody (R&D Systems), MECA-32 rat anti-mouseendothelial monoclonal antibody (CD Pharmingen), mouse anti-human CD34monoclonal antibody, anti-human CD31 monoclonal antibody, and rabbitanti-human von Willebrand factor (Dako, Carpinteria, Calif.) wereemployed. After overnight incubation with the primary antibodies at 4°C., detection was performed using biotinylated donkey secondaryantibodies (Jackson ImmunoResearch, West Grove, Pa.), followed bystreptavidin-Alexa488 conjugate (Invitrogen, Carlsbad, Calif.).Propidium iodide (1 μg/ml) was used as a counterstain for the nuclei.For immunostaining of the Matrigel™ plugs, 7 μm cryosections wereprepared and fixed with 4% paraformaldehyde (PFA) for Hematoxylin andEosin (H&E) staining and immunohistochemistry (IHC). Incubation withprimary antibody was performed as described above. Bound antigen wasdetected with donkey biotinylated anti-rabbit secondary antibody,followed by streptavidin-HRP (both from Jackson ImmunoResearch) and DABor AEC.

Microarray Gene Expression Profile.

Affymetrix's Human Genome U133 Plus 2.0 Array was used to analyze geneexpression. Total RNA was extracted from control ECs andE4ORF1-expressing ECs using Trizol™ (Invitrogen). ECs were grown for 24hours under serum-free, growth factor-free medium prior to RNAextraction. cDNA was synthesized from the total RNA extracted.Biotinylated cRNA was made from the cDNA by in vitro transcription usinga kit (Enzo Diagnostic). Fragmented cRNA was hybridized to the genechips, washed, and stained with streptavidin phycoethyrin. The probearrays were scanned with the Genechip System confocal scanner, dataprocessed with Affymetrix Microarray Suite 4.0 and analysis wasperformed using Genespring GX (Agilent).

Adenovirus E4ORF1 Promotes Survival of Endothelial Cells

Adenovirus E4 positive vectors that express all six E4 gene productspromote EC survival and prevent apoptosis even in of the absence ofpro-angiogenic factors, including VEGF-A and serum (Zhang et al. (2004)J Biol Chem 279(12):11760-66). However, prior to this invention, it wasnot clear which of the six E4 open reading frame (ORF) genes of the E4gene confers this unique pro-angiogenic phenotype to the ECs. Toidentify the specific adenoviral E4ORF, which selectively promotesneo-angiogenesis, ECs were infected in serum-free, growth factor-freemedium with adenoviral vectors carrying various E4ORF sequencedeletions, including AdE4 vectors lacking either E4ORF1, E4ORF4, E4ORF6,or a combination of E4ORF3,4,6,6/7. As shown in FIGS. 1A and B, anincrease in the survival of ECs was observed in cells infected withE4ORF1, including AdE4ORF1→7, which expresses all six E4 ORFs andAdE4ORF1, which only expresses E4ORF1. In contrast, AdE4ORF3,4,6,6/7,AdE4ORF4 and AdE4ORF6 did not improve EC survival in serum-free, growthfactor-free conditions. Therefore, the invention provides that among theknown E4ORFs that are known to convey survival signals, E4ORF1 plays acentral role in the AdE4 regulation of EC survival.

In order to rule out the possible involvement of other adenovirus genesin the E4ORF1 effect, a lentivirus specifically expressing only E4ORF1without any other adenoviral genes was generated. ECs were infected withLenti-GFP or Lenti-E4ORF1. Infection with the E4ORF1-lentivirussignificantly increased the survival of ECs in serum-free, growthfactor-free medium compared to infection with GFP-lentivirus or tonon-infected (control) ECs (FIGS. 1C and D). Transfection efficiency ofECs was almost 100%, as measured by GFP expression in Lenti-GFP infectedcells. A similar effect of E4ORF1-lentivirus was also seen in lymphaticendothelial cells (FIG. 1E). In summary, the E4ORF1-lentivirusreplicated the AdE4ORF1 EC survival effect, including the typicalcobblestone morphology of ECs grown in monolayers. These data furtherindicate that E4ORF1 is responsible for the pro-survival effect of AdE4in ECs.

To further verify the ability of E4ORF1 to selectively promote theoutgrowth of organ-specific endothelial cells, crude populations ofenzymatically digested human testicular cells or fresh human umbilicalcord blood mononuclear cells were infected with Lenti-E4ORF1. Comparedwith non-infected cultures, ECs infected with the E4ORF1-lentivirussupported the outgrowth of monolayers of these organ specific ECs (FIG.1F). The ECs were highly pure, grew in a contact inhibited manner, andexpressed the typical markers of the endothelium, including CD34, vonWillebrand factor, VE-cadherin, and CD31 (FIG. 8). These data furthersupports the finding that E4ORF1 selectively confers pro-survivaleffects to organ-specific ECs.

E4ORF1 Activates Phosphorylation of Akt

Phosphorylation of Akt is a key component of AdE4ORF1→7-mediated ECsurvival (Zhang et al. (2004) J Biol Chem 279(12):11760-66). To testwhether E4ORF1 is responsible for the phosphorylation of Akt inAdE4ORF1→7 transduced ECs, siRNA against E4ORF1 was used to inhibit theexpression of E4ORF1. Infection of the ECs with either AdE4ORF1→7 orAdE4ORF1, but not AdE4ORF3,4,6,6/7, induced phosphorylation of Akt atSer473, with total Akt remaining constant (FIG. 2A). However,pretreatment of ECs with siRNA against E4ORF1 completely inhibitedAdE4ORF1→7 virus-induced pAkt activation, as compared to the ECstransfected with either fluorescein-labeled siRNA (control) or E4ORF6siRNA (FIG. 2B). Moreover, infection of ECs with the lentiviral E4ORF1vector also increased Akt phosphorylation at Ser-473 (FIG. 2C) comparedto control or to infection with Lenti-GFP, indicating that Aktactivation is E4ORF1-specific. Infection of smooth muscle cells withlentiviral E4ORF1 had no significant effect on Akt activation, showingthat E4ORF1's ability to phosphorylate Akt is specific to ECs.

To test whether AdE4ORF1 prolongs EC survival through mTOR or PI3Ksignaling, AdE4ORF1-infected ECs were exposed to rapamycin, an inhibitorof mTOR signaling, or to LY-294002, an inhibitor of PI3K signaling. Thepro-survival effects of AdE4ORF1 were only moderately reduced byrapamycin (FIG. 2D). In comparison, LY-294002 significantly suppressedthe effect of AdE4ORF1, and LY-294002 together with rapamycin completelyeliminated the E4ORF1 survival effect (FIG. 2D). Together, these resultsindicate that E4ORF1-mediated survival, like AdE4-mediated survival,involves EC-specific Akt activation, and that the mTor-PI3K signalingpathway is also likely involved. To determine whether E4ORF1 prolongedEC survival was regulated by MAPK, E4ORF1-infected ECs were treated withMAPK inhibitors PD98059, a selective inhibitor of ERK, and SB203580, aselective inhibitor of p38-MAPK). Neither PD98059 nor SB203580 hadsignificant effects on the survival effects of E4ORF1-expressing ECs.These results indicate the E4ORF1 survival effect involves theEC-specific Akt activation and in part is mediated through therecruitment of the PI3K-Akt-mTOR signaling pathway.

E4ORF1 Induces Expression of FGF-2

In order to identify the mechanism by which AdE4ORF1 promotes thepro-angiogenic effect on ECs, a microarray expression analysis wasperformed on Lenti-E4ORF1-infected ECs and compared them to theLenti-GFP infected ECs, both grown under serum-free, growth factor-freeconditions for 24 hours. The commonly known pro-angiogenic factorsessential for survival and proliferation of ECs maintained theirexpression pattern in both E4ORF1-infected and GFP-infected ECs. Resultsare shown in FIG. 10. This data indicates that although naïve ECsrequire exogenous FGF-2 for optimal activation, E4ORF1-infected ECsrequires Akt-dependent intrakine activation of the FGF-2/FGF-R1 pathwayfor survival.

Whether AdE4ORF1→7 or AdE4ORF1 (expressing only E4ORF1) promotes ECsurvival via increased expression of the pro-angiogenic factor FGF-2 wasnext examined. As shown in FIGS. 3A and 3B, ECs infected with AdE4ORF1or AdE4ORF1→7 significantly increased FGF-2 protein expression whencompared to naïve ECs or ECs infected with adenoviral vectors lackingE4ORF1. ECs infected with Lenti-E4ORF1 also increased protein levels ofFGF-2 (FIG. 3B). FGF-2 knockdown by FGF-2 siRNA blocked E4ORF1-mediatedsurvival (FIGS. 3C and D). Diminished FGF-2 protein expression by theFGF-2 siRNA was confirmed by Western blot analysis and suppressedE4ORF1-induced FGF-2 protein expression (FIG. 3B).

In addition to the FGF-2/FGFR-1 pathway, VEGF-A/VEGF-R could alsocontribute to the E4ORF1-mediated survival effect seen in ECs. AlthoughSU5402, an inhibitor of FGF-R1 tyrosine kinase activity, blocked thisexpansion of rFGF-2-treated E4ORF1-infected ECs, the VEGF-A receptorinhibitor SU5416 did not significantly affect the survival orproliferation of E4ORF1-expressing ECs, either in the presence orabsence of FGF-2 (FIG. 3E). Taken together, this shows that E4ORF1exerts its pro-survival effect in part through the activation of theFGF-2/FGFR-1 complex rather than the VEGF-A/VEGFR pathway.

To further attribute the pro-survival effect of E4ORF1-expressing ECsthrough the activation of FGFR-1, recombinant FGF-2 (rFGF-2) was addedto the serum-free culture medium (FIG. 3F). In serum-free, growthfactor-free medium, E4ORF1-expressing ECs survived without anyproliferation, maintaining their contact-inhibited status, whereas ECswhich did not express the E4ORF1 gene product rapidly underwentapoptosis. The addition of rFGF-2 to serum-free, growth factor-freecultures of E4ORF1-expressing ECs robustly increased the proliferationof ECs. rFGF-2 briefly supported the survival of ECs which did notexpress E4ORF1, but these cells eventually became progressivelyapoptotic. Addition of exogenous recombinant VEGF-A (rVEGF-A) alone orwith rFGF-2-treated E4ORF1-expressing ECs had a weak effect on survivalor expansion of ECs expressing E4ORF1, as compared to treatment withrFGF-2 alone. These data shows that E4ORF1-mediated upregulation of theFGF-2/FGF-receptor system plays a critical role for the E4ORF1-mediatedsurvival of ECs, while addition of exogenous FGF-2 not only promotessurvival, but also induces proliferation of E4ORF1-expressing ECs inserum-free, growth factor-free conditions.

E4ORF1 Maintains the Functional Properties of ECs.

ECs have the capacity to carry out angiogenic processes in vitro,including the ability to form tubes and to migrate. WhetherE4ORF1-infected ECs retain these properties was next examined. As shownin FIG. 9, infection with AdE4ORF1 enhanced the wound-induced migrationof ECs in serum-free, growth factor-free medium. This effect is similarto that observed when exogenous rFGF-2 is added to ECs. In contrast,both uninfected and AdE4^(null)-infected ECs in serum-free, growthfactor-free medium were unable to migrate, instead undergoing apoptosis.Whether E4ORF1 affects EC tube formation was investigated. ECs wereinfected with AdE4ORF1 or AdE4ORF3,4,6,6/7 for 24 hours before beingseeded in Matrigel™. As shown in FIGS. 9B and 9C, ECs infected withAdE4ORF1 or Lenti-E4ORF1 maintained their ability to form tubes inserum-free conditions for over 2 days, whereas control ECs orAdE4ORF3,4,6,6/7-infected ECs only maintained tube-forming activity forless than 1 day, after which there was a rapid dissociation of thetubes. These data indicate that E4ORF1-infected ECs maintainneo-angiogenic potential.

E4ORF1-Expressing ECs Rapidly Organize into Vessel-Like Structures InVivo

Primary human endothelial cells have limited survival and functionalityin mouse xenograft angiogenesis assays (Koizumi et al. (2003) BiochemBiophys Res Comm 306:219-24). Based on the phenotypic changes observedin vitro in human ECs stably transduced with lenti-E4ORF1 that impliedan enhanced angiogenic capability, the performance of these cells invivo was next assessed. Whereas normal ECs were barely detectable fivedays after injection subcutaneously into immunocompromised mice (FIG.4A), E4ORF1-expressing EC survived and established vascular nodules(FIG. 4B), and organized rapidly into primitive vascular-type structures(FIGS. 4C and D). These exhibited circular morphology in cross-section,with a central core of degenerating red blood cells. To distinguishhuman EC from host (mouse) endothelium, species-specific antibodies wereemployed and unequivocal labeling of nests of engrafted ECs withanti-human VE-cadherin was observed (FIG. 4E), which did not recognizeadjacent mouse vessels. Conversely, labeling of mouse endothelium withMECA-32 delineated the adjacent mouse vessels surrounding but notintermingling with AdE4ORF1 ECs (FIG. 4F). Consistent with thebiochemical profile of AdE4ORF1-infected ECs in vitro, robust FGF-2expression was detected in the engrafted tissue compared to the adjacenthost connective tissue (FIG. 4G). Staining of these sections with aphospho-Akt specific antibody shows that E4ORF1-expressing ECs hadmaintained their chronic Akt activation (FIG. 4H). These data indicatethat for at least five days following Matrigel™ implantation,E4ORF1-expressing ECs retained their Akt-dependent neo-angiogenicpotential, forming vascular structures without generating tumorigenicvessels.

In order to determine the capacity of E4ORF1-expressing ECs to assembleinto functional vessels in the Matrigel™ implants in vivo, monolayers ofE4ORF1 ECs (transduced by Lenti-E4ORF1) or naïve ECs were labeled withLenti-GFP. Ten million of either cell type were inoculated withMatrigel™ into the flanks of NOD-SCID mice. Vessels were given two weeksto assemble in the plugs at which point, the mice were injectedintravenously with human endothelium-specific UEA-1 lectin to examinefunctionality of the vessels and the presence of human derivedGFP-positive functional neo-vessels within the Matrigel™. The resultswere evaluated by stereo confocal microscopy. Similar to naïve ECs(FIGS. 5A and C), ECs expressing E4ORF1 (FIGS. 5B and D) were able toassemble into functional, branching GFP⁺, lectin⁺ neo-vessels connectingto the host vessel (FIGS. 5C and D). ECs expressing E4ORF1 also gaverise to long sprouting GFP⁺ lectin⁺ neo-vessels (FIG. 5E). These datasuggest that E4ORF1-expressing ECs maintain their angiogenic repertoireand can assemble into neo-vessels, even in the absence of exogenousgrowth factors.

E4ORF1-Expressing ECs can Assemble into Neo-Vessels in LeukemicXenografts

As an alternative means to assess the pro-angiogenic properties ofE4ORF1-expressing EC in vivo, human HL60 leukemia cells were combinedwith E4ORF1-expressing ECs in NOD-SCID mice subcutaneously. Whereasnormal ECs do not survive within human tumor xenografts, the humanVE-cadherin⁺ E4ORF1 ECs not only survived for at least 10 days withinthe HL60 tumors, but also formed vessel-like structures with a lumen(FIGS. 5F and G). Double labeling for mouse (MECA-32) and human(VE-cadherin) endothelium confirmed not only the species specificity ofthe antibodies but also revealed formation of E4ORF1-expressing vesselswithin the tumor. These data reveal the propensity of E4ORF1 to induce apro-angiogenic phenotype in endothelial cells in vivo in either a benignsetting or in tumors.

E4ORF1 Expression in ECs Supports the Expansion of Leukemic Cells inSerum-Free, Growth Factor-Free Conditions

To determine whether ECs in serum-free, growth factor-free conditionscan induce proliferation in leukemic HL60 cells, HL60 cells were grownin serum-free, cytokine-free conditions as well as in co-culture withE4ORF1-expressing ECs. In serum-free, cytokine-free conditions, HL60cells underwent apoptosis (FIG. 5I). However, culture of the HL60 cellswith E4ORF1 ECs in serum-free, cytokine-free conditions resulted in aremarkable expansion of HL60 cells (FIG. 5I). During the culture period,E4ORF1 ECs maintained their integrity, facilitating proliferation of theleukemic cells. This demonstrates the ability of ECs to support theexpansion of leukemic cells without addition of exogenous cytokines.

Discussion

E4 gene of the adenoviral vector plays an important role in promotingsurvival of ECs and regulating angiogenic potential of ECs by recruitingthe VE-cadherin/Akt-signaling cascade. Remarkably, this pro-survivaleffect of the E4 gene is restricted to vascular cells but notmesenchymal cells. The specific E4ORF gene that enhances thepro-angiogenic effect of the ECs has been identified. Among the sixpotential gene products transcribed by the E4ORFs of adenoviral E4 gene,the E4ORF1 gene potentiates the survival and migration of ECs, both invitro and in vivo. The pro-angiogenic effect of E4ORF1 conferred to ECsis mediated through the activation of PI3K-mTOR and Akt signalingpathway. Remarkably, inhibition of mTOR is highly effective in blockingpro-angiogenic effect afforded by the E4ORF1 gene. Moreover, FGF-2expression is increased by infection with E4ORF1-infected ECs leading tostimulation of angiogenesis both in vivo and in vitro. These resultsshow that the E4ORF1 protein augments the survival of ECs throughrecruiting FGF-2 and activation of pro-angiogenic signaling pathways.

In addition to maintaining the angiogenic effect of ECs,E4ORF1-expressing ECs can also contribute to angiogenesis in leukemicchloromas. ECs infected with E4ORF1 supported long term expansion ofleukemic cells under serum-free, growth factor-free conditions, withoutthemselves undergoing apoptosis. Thus, the survival and angiogeniceffects of ECs expressing E4ORF1 were sustained without immortalizationof such cells. This has allowed the culture of pure populations oforgan-specific ECs from testis and umbilical cord blood. Thus, E4ORF1facilitates the isolation of intact organ-specific endothelial cells toexamine their potential in regulating vascularization of regeneratingorgans, and identify the mechanism by which ECs contribute to tumorneovascularization.

Adenovirus gene transfer vectors carrying the full compliment of the E4gene activates PI3K in ECs and other cell types (Zhang et al. (2004) JBiol Chem 279(12):11760-66; Rajala et al. (2005) J Virol79(19):12332-41; Liu et al. (2005) J Virol 79(23):14507-15). Theinvention provides that E4ORF1 protein is an essential product foradenovirus E4-mediated activation of PI3K. Activating PI3K by E4ORF1resulted in increased RhebGTP loading and stimulating mTOR activity(O'Shea et al. (2005) Embo J 24(6):1211-21). PI3K/mTOR signaling pathwayseem to play a critical role in mediating survival, replication, andmigration of mammalian cells (Brunet et al. (2001) Curr Opin Neurobiol11(3):297-305). Inhibition of mTOR by rapamycin only partially reversedthe E4ORF1 effect of promoting EC survival, indicating that mTOR may notbe a unique signal pathway for the E4ORF1 effect (FIG. 2B). Indeed, theinvention provides that E4ORF1 increased FGF-2 synthesis in EC.Activation of Src and PI3K-Akt signaling may target in parallel withFGF-2 expression in an mTOR-independent manner (Zhang et al. (2006) JBiol Chem 281(2):905-14). Thus, the results indicate that E4ORF1 drivesEC survival and angiogenesis via mTOR-dependent and an additionalmTOR-independent signal pathway.

Exposure of survival factors, such as FGF-2 and VEGF-A, is important formaintaining EC survival and migration (Meredith et al. (1993) Mol BiolCell 4(9): 953-61). These growth factors have been shown to be importantin initiating neo-angiogenesis (Veikkola et al. (2000) Cancer Res60(2):203-12; Seghezzi et al. (1998) J Cell Biol 141(7): 1659-73).E4ORF1 product has been shown in this invention to increase FGF-2expression in EC monolayers. However, significant changes of VEGF-Aexpression in ECs were not detectable by ELISA and Western blot. Thus,the effect of E4ORF1 on the angiogenic process can result from anincrease in endogenous FGF-2 expression. FGF-2 serves both paracrine andautocrine functions in the regulation of angiogenesis. Overexpression ofendogenous FGF-2 in vivo has significant pro-angiogenic effect,increasing capillary density and improving cardiac resistance to injury(Sheikh et al. (2001) Am J Physiol Heart Circ Physiol280(3):H1039-1050), whereas FGF-2 knockout mice exhibit significantdelay in the rate of healing of full-thickness excisional skin wounds(Ortega et al. (1998) Proc Natl Acad Sci USA 95(10):5672-7). Studieshave shown that FGF-2 is a downstream target of PI3K in many importantcellular pathways (Zhang et al. (2006) J Biol Chem 281(2):905-14; Weylieet al. (2006) J Vasc Res 43(1):61-9). However, FGF-2 can also activatePI3K/Akt signaling pathway (Vandermoere et al. (2005) Oncogene24(35):5482-91). Thus, FGF2-activated Akt pathway may involve a positivefeedback loop. Indeed, E4ORF1 activated both PI3K and FGF-2 expression,and thus, exerting a synergistic effect on EC survival and angiogenesis.

Adenovirus E4ORF1 has been identified interacting with several differentcellular PDZ proteins, including MAGI-1, MUPP1 and ZO-2 (Glaunsinger etal. (2000) Oncogene 19(46):5270-80; Songyang et al. (1997) Science275(5296):73-7). PDZ proteins regulate cell growth, polarity, andadhesion in response to cell contact (Craven and Bredt (1998) Cell93(4):495-8; Fanning and Anderson (1999) J Clin Invest 103(6):767-72).MAGI-1 formation of complexes with B-catenin and E-cadherin mightcontribute to the formation of adherens molecular complexes at tightjunctions in endothelial and epithelial cells (Wegmann et al. (2004) ExpCell Res 300(1):121-33; Dobrosotskaya and James (2000) Biochem BiophysRes Commun 270(3):903-9). Overexpression of the N-terminal domain ofZO-1 in connexin 43-expressing cells resulted in redistribution ofconnexin 43 from cell-cell interfaces to cytoplasmic structures(Toyofuku, et al. (1998) J Biol Chem 273(21):12725-31). Consistent withthese results, it was demonstrated that AdE4 activated theVE-cadherin/Akt pathway and modulated connexin 40 and 43 expression(Zhang et al. (2004) J Biol Chem 279(12):11760-66; Zhang et al. (2005)Circ Res 96(9):950-7).

E4ORF1 activates PI3K and FGF-2 pathways to enhance the survival andinduce neo-angiogenesis in ECs. These findings show that the E4ORF1product possesses a potential novel role for AdE4 in regulation ofangiogenesis and survival of ECs in vitro and in vivo. In one aspect ofthe invention, E4ORF1 can be used as an intracellular decoy to delineatepro-angiogenic pathways mediating assembly of new vessels. As such,infection of E4ORF1 can be exploited to induce revascularization of theischemic tissues.

Virulent adenoviruses are evolved to hijack the molecular machinery ofthe host cells, such as endothelial cells, to replicate the viralgenome, while keeping the host cell alive. The discovery of thisinvention has taken advantage of the capacity of adenoviral E4 genecomplex to activate cell survival signals, in order to identify geneproducts that can preserve long-term endothelial cell integrity. Amongthe six gene products transcribed by the adenoviral E4 gene complex, theinvention provides the discovery that the E4ORF1 gene product is the keygene that endows endothelial cells with the capacity to survive andproliferate in a contact-inhibited manner and preserves theirneo-angiogenic potential, all in the absence of serum or endothelialcell growth factor supplements. E4ORF1-expressing ECs maintained thecomplete angiogenic profile of ECs and assembled into functionalneo-vessels in vitro and in vivo and contributed to angiogenesis inleukemic chloromas. Although naïve ECs underwent apoptosis within 6hours in cytokine- and serum-free conditions, E4ORF1-expressing ECssupported long-term expansion of leukemic cells under serum- andcytokine-free conditions, without themselves undergoing apoptosis.Through tonic stimulation of PI3K-Akt-mTOR activating the FGF-2/FGFR-1pathway, E4ORF1-expressing ECs supported the sustenance of theangiogenic phenotype without immortalization. This approach has alsopermitted the culture of pure populations of organ-specific ECs fromtestis and umbilical cord blood. As such E4ORF1 facilitates isolation ofintact organ-specific endothelial cells to examine their potential inregulating vascularization of regenerating organs, and to define themechanisms by which endothelial cells contribute to tumorneo-angiogenesis. Importantly, this enables for the first time the useof minimal media conditions (i.e., exogenous cytokine- and serum-freemedia) in the assay of endothelial-derived signals that support canceror normal stem cell self-renewal.

Evidence indicates that organ-specific endothelial cells are not onlyconduits for delivery of oxygen and nutrients, but also they provide apermissive environment for the survival and growth of organ-specificstem and progenitor cells (Rafii et al. (1995) Blood 86:3353-63; Lammertet al. (2001) Science 294:564-7; Rafii, S., et al. (1994) Blood84:10-19; Kiel et al. (2005) Cell 121:1109-21; Avecilla et al. (2004)Nat Med 10:64-71; Yoshida et al. (2007) Science 317:1722-26; Nikolova etal. (2006) Dev Cell 10:397-405; Dias et al. (2001) Proc Natl Acad SciUSA 98:10857-62; Gilbertson & Rich (2007) Nat Rev Cancer 7:733-6;Calabrese et al. (2007) Cancer Cell 11:69-82). Based on primarily invivo studies, ECs have been shown to elaborate membrane or solublecytokines (Rafii et al. (1995) Blood 86:3353-63; Avecilla et al. (2004)Nat Med 10:64-71; Yoshida et al. (2007) Science 317:1722-26; Dias et al.(2001) Proc Natl Acad Sci USA 98:10857-62; Calabrese et al. (2007)Cancer Cell 11:69-82) or deposit unique matrix (Nikolova et al. (2006)Dev Cell 10:397-405) that contribute to the specification anddifferentiation of organ-specific stem cells. The use of immortalizedendothelial cells lines has manifested marginal benefit in elucidatingthe factors that support tumorigenesis. In addition, culture of ECs, inthe absence of an enriched cocktail of pro-angiogenic factors results inthe apoptosis of ECs within a few hours, precluding further study.Indeed, the contribution of ECs to stem and progenitor cell biology hasbeen obscured by the presence of stem cell-active cytokines, includingFGFs, VEGFs, TGF-β and EGFs, which can by themselves promote thesurvival of stem and tumor cells. In fact, it is not clear in previouspublished studies (Rafii et al. (1995) Blood 86:3353-63; Nikolova et al.(2006) Dev Cell 10:397-405; Shen et al. (2004) Science 304:1338-40)whether proliferation of stem cells was mediated by elaboration offactors by endothelial cells or by exogenously added FGFs, and EGFs.Therefore, the unique capacity of the compositions and methods of theinvention to maintain the neo-angiogenic state of ECs without theotherwise ubiquitous requirement for numerous exogenous cytokinesprovides for a radically different in vitro platform to identify andcharacterize the factors that promote the growth of stem and tumorcells. In addition, introduction of the E4ORF1 compositions of theinvention allows for purification of organ specific endothelial cells.To date, isolation of endothelial cells from human and mouse tissues,including testis and lung has been achieved. These findings provide atechnical advance in the study of the heterogeneity of endothelial cellswithout compromising their neo-angiogenic integrity.

The ability to induce neo-angiogenesis by E4ORF1 and the compositions ofthe invention without oncogenic transformation is a unique feature ofthe E4ORF1 gene product that is primarily conferred to the humanendothelial cells. E4ORF1 cloned from either type 5 or 9 adenoviruseshad no oncogenic effect, and E4ORF1-expressing ECs maintained bothcontact inhibition and the requirement for exogenous FGF-2 toproliferate. The pro-angiogenic effect of E4ORF1 conferred to ECs wasmediated through the activation of PI3K-mTOR and Akt signaling pathway.Inhibition of mTOR was highly effective in blocking the pro-angiogeniceffect afforded by the E4ORF1 gene. Moreover, FGF-2 expression wasincreased by expression of E4ORF1 in ECs, leading to stimulation ofangiogenesis both in vivo and in vitro. These results suggest that theE4ORF1 protein augments the survival of ECs by activation ofFGF-2/FGFR-1 pro-angiogenic signaling pathways without oncogenictransformation.

This invention provides the finding that E4ORF1 can induce tonicactivation of Akt. This is important, since naive ECs require chronicstimulation with a wide variety of growth factors, including VEGF-A,FGF-2 and EGF, in order to maintain activation of Akt and cellproliferation. In fact, VEGF-A or FGF-2 stimulation of naïve ECs resultsin phosphorylation of Akt for only 10 minutes, with rapiddephosphorylation and activation of the apoptotic pathway, if the cellsare not stimulated again with growth factors. However, E4ORF1-expressingECs maintained Akt phosphorylation indefinitely, showing that E4ORF1gene product can interfere with regulatory pathways to deactivate Aktor, alternatively, can induce constant activation of Akt through a novelrecruitment of the PI3K-mTOR pathway. As rapamycin was only partiallyeffective in reversing the pro-survival effect of E4ORF1, these datashow that other pathways drive E4ORF1-activation of Akt. Akt-mediatedupregulation of FGF-2 can contribute to persistent activation of PI3Kthrough an intrakine FGF-2/FGFR-1 activation.

The mechanism by which E4ORF1 activates Akt is complex and involves aninteraction with cytoskeletal regulatory elements. E4ORF1 isolated fromadenovirus type 9, which has primarily been studied in mouse stromalcells, has been found to interact with several different cellular PDZproteins, including Dlg1, MAGI-1, MUPP1 and ZO-2 (Glaunsinger et al.(2000) Oncogene 19:5270-80; Frese et al. (2003) Oncogene 22:710-21). PDZproteins regulate cell growth, polarity, and adhesion in response tocell contact. In mouse stromal cells, specific binding of E4ORF1 toendogenous Dlg1 promotes Ras-mediated PI3K activation (Frese et al.(2003) Oncogene 22:710-21). Formation of complexes containing MAGI-1,β-catenin, and E-cadherin can contribute to the formation of adherensmolecular complexes at tight junctions in endothelial and epithelialcells. All together, activation of this cascade of events could resultin chronic activation of Akt without cellular transformation. Consistentwith these results, it was demonstrated that AdE4 activated theVE-cadherin/Akt pathway and modulated connexin 40 and 43 expression(Zhang et al. (2004) J Biol Chem 279:11760-6; Zhang et al. (2005) CircRes 96:950-7). Therefore, interaction of E4ORF1 protein and PDZ can playan initial role in regulation of survival and angiogenesis in human ECs,as well.

An advantage of the invention is reproducible maintenance of theangiogenic state of the E4ORF1-expressing ECs. This permanent and robustangiogenic state, which has never been reported by other genetransduction approaches is most likely due to persistent activation ofAkt. Remarkably, the physiological effect of E4ORF1 was pronounced inendothelial but not smooth muscle, hematopoietic cells or even tumorcells. Indeed, numerous studies have shown that activation of Akt playsa critical role in modulation of the angiogenic state in endothelialcells (Chen et al. (2005) Nat Med 11:1188-11; Ackah et al. (2005) J ClinInvest 115:2119-27; Phung et al. (2006) Cancer Cell 10:159-70; Mukai etal. (2006) J Clin Invest 116:334-43; Phung et al. (2007) Cancer Res67:5070-5; Somanath et al. (2007) J Biol Chem 282:22964-76). The extentof Akt activation dictates the level of angiogenic activity.Over-expression of Akt results in induction of neo-angiogenesis,vascular leakiness, and enhanced tumor growth (Phung et al. (2006)Cancer Cell 10:159-70). In contrast, absence of Akt1 may affect thestability and patterning of neo-angiogenesis (Ackah et al. (2005) J ClinInvest 115:2119-27). Therefore, the chronic low level activation of Aktafforded by E4ORF1 provides the precise signals to confer toE4ORF1-expressing ECs a long-lasting angiogenic state, withoutdysregulated growth.

Exposure to survival factors, such as FGF-2 and VEGF-A, is important formaintaining EC survival and migration (Rafii et al. (1995) Blood86:3353-63). These growth factors have been shown to be important ininitiating neo-angiogenesis. Here, the E4ORF1 product increased FGF-2expression, survival and proliferation of EC monolayers. VEGF-A has onlya marginal effect on increasing proliferation of E4ORF1-expressing ECs.The effect of E4ORF1 on the angiogenic process can result from anincrease in endogenous FGF-2 expression. FGF-2 serves both paracrine(Chung et al. (2007) J Virol 81:4787-97) and autocrine functions in theregulation of angiogenesis. Overexpression of endogenous FGF-2 in vivohas a significant pro-angiogenic effect, increasing capillary densityand improving cardiac resistance to injury. FGF-2 is a downstream targetof PI3K in many important cellular pathways (Zhang et al. (2006) J BiolChem 281:905-14; Weylie et al. (2006) J Vasc Res 43:61-69). FGF-2 canalso activate the PI3K/Akt signaling pathway. The FGF-2-activated Aktpathway can involve a positive feedback loop. E4ORF1 activated both PI3Kand FGF-2 expression, thus exerting a synergistic effect on EC survivaland angiogenesis.

The E4ORF1 adenoviral gene, through chronic activation of theFGF-2/PI3K-Akt-mTOR pathway, enhanced survival, and maintainedneo-angiogenesis in ECs without cellular transformation. The E4ORF1 geneproduct supported survival of the ECs in the absence of growth factorsand serum but exogenous stimulation with FGF-2 was still needed toinduce contact inhibited proliferation. The compositions of theinvention can hijack pro-angiogenic signals of the host endothelium andenhance their invasiveness and establish their pathological damage tothe infected tissues. In addition, E4ORF1 organ specific EC culturesprovide methods to decipher the unique molecular and cellular pathwaysactivated in neo-angiogenic endothelium that support organogenesis andtumorigenesis.

Example 2 E4ORF1-Expressing Feeder Cells

Stem and progenitor cells are notoriously difficult to maintain and growin culture. Many such cells must be grown with “feeder cells”. Forexample, feeder layers comprising primary endothelial cells can be usedto support the growth of hematopoietic stem cells. However, these feederlayers of primary endothelial cells generally require the presence ofserum or growth factors for long-term maintenance. Many stem cellscannot tolerate the presence of serum or certain growth factors.Experiments were performed to determine whether E4ORF1-expressingendothelial cells would be useful as feeder cells to support the growthof stem cells and progenitor cells such as hematopoietic stem orprogenitor cells. FIG. 6 provides a graph showing that a feeder celllayer of E4ORF1-transduced endothelial cells induced expansion of CD34+hematopoietic progenitor cells over a five-week period, even in theabsence of serum.

1.-85. (canceled)
 86. A composition comprising a population endothelialcells, wherein the endothelial cells (a) comprise a recombinant nucleicacid sequence that comprises an E4ORF1 coding sequence under the controlof a promoter, and (b) do not comprise nucleic acid sequences thatcomprise the entire adenovirus E4 region.
 87. The composition of claim86, wherein the recombinant nucleic acid sequence is comprised within alentiviral vector.
 88. The composition of claim 86, wherein therecombinant nucleic acid sequence is comprised within an adenoviralvector.
 89. The composition of claim 86, wherein the recombinant nucleicacid sequence further comprises an antibiotic resistance gene.
 90. Thecomposition of claim 86, wherein the recombinant nucleic acid sequencefurther comprises a reporter gene or a sequence encoding an expressiontag.
 91. The composition of claim 90, wherein the recombinant nucleicacid sequence encodes green fluorescent protein (GFP).
 92. Thecomposition of claim 86, wherein the promoter is selected from the groupconsisting of a CMV promoter, an SV40 promoter, an RSV promoter, anHIV-Ltr promoter, and an MML promoter.
 93. The composition of claim 86,wherein the promoter is an adenovirus promoter.
 94. The composition ofclaim 86, wherein the promoter is an inducible promoter.
 95. Thecomposition of claim 86, wherein the E4ORF1 coding sequence is a humanadenovirus type 5 coding sequence.
 96. The composition of claim 86,wherein the E4ORF1 coding sequence comprises SEQ ID NO.
 1. 97. Thecomposition of claim 86, wherein the E4ORF1 coding sequence is an E4ORF1coding sequence from human adenovirus 1, human adenovirus 2, humanadenovirus 3, human adenovirus 7, human adenovirus 9, human adenovirus11, human adenovirus 12, human adenovirus 14, human adenovirus 34, humanadenovirus 35, human adenovirus 46, human adenovirus 50, or humanadenovirus
 52. 98. The composition of claim 86, where the endothelialcells are selected from the group consisting of: testis endothelialcells, lung endothelial cells, lymphatic endothelial cells, cord bloodendothelial cells, umbilical vein endothelial cells, heart endothelialcells, skin endothelial cells, liver endothelial cells, brainendothelial cells, bone endothelial cells, and pancreatic endothelialcells.
 99. The composition of claim 86, further comprising a populationof cancer cells, stem cells, or progenitor cells.
 100. The compositionof claim 99, wherein the stem cells or progenitor cells are selectedfrom the group consisting of embryonic stem cells, hematopoietic stem orprogenitor cells, neural stem or progenitor cells, skin stem orprogenitor cells, spermatogonial stem or progenitor cells, and gut stemor progenitor cells.
 101. The composition of claim 86, furthercomprising a population of hematopoietic stem or progenitor cells. 102.The composition of claim 86, further comprising Matrigel.
 103. Thecomposition of claim 86, further comprising a physiological salinesolution or a carrier solution suitable for administration to a livingsubject.
 104. A method of inducing angiogenesis in vivo, the methodcomprising administering a composition according to claim 86 to asubject in need thereof.
 105. A method of inducing wound healing invivo, the method comprising administering a composition according toclaim 86 to a subject in need thereof.
 106. A method for maintaining orexpanding cancer cells, stem cells or progenitor cells in vitro, themethod comprising: a. contacting cancer cells, stem cells or progenitorcells in vitro with conditioned medium, wherein the condition medium isderived from a culture of E4ORF1-expressing endothelial cells that donot contain or express nucleic acid sequences that comprise the entireadenovirus E4 region, thereby maintaining or expanding the cancer cells,stem cells or progenitor cells in vitro.
 107. A method of maintaining orexpanding cancer cells, stem cells or progenitor cells in vitro, themethod comprising: b. culturing a population of cancer cells, stem cellsor progenitor cells in vitro in a culture vessel that comprises apopulation of E4ORF1-expressing endothelial cells, wherein theendothelial cells do not contain or express nucleic acid sequences thatcomprise the entire adenovirus E4 region, thereby maintaining orexpanding the cancer cells, stem cells or progenitor cells in vitro.